Stem cells refer to cells that can proliferate indefinitely in an undifferentiated state as well as differentiate into certain specific cell types under the effect of appropriate stimuli or environments. Since human pluripotent stem cells are able to proliferate indefinitely under in vitro culture conditions (self-renewal) and differentiate into nearly all cell types of an individual (pluripotency), findings in the studies of human pluripotent stem cells are utilized in a wide variety of fields, including basic studies for understanding the development, differentiation and growth of an individual, development of cell therapy products for the treatment of damages or various diseases of an individual, and efficacy screening for candidate therapeutic drugs, disease etiology study, and development of therapy strategies. Even though the demand for human pluripotent stem cells is rapidly increasing in a variety of fields, there is an obstacle to the use of such cells in the development of the related technology because culture media for maintenance and culture of undifferentiated human pluripotent stem cells are limited and the culture of pluripotent stem cells is difficult and laborious. In particular, for the development of cell therapy, it is necessary to establish clinically applicable culture conditions such as serum-free medium devoid of animal-derived products and develop large-scale culture systems capable of supplying them at a sufficient amount when needed.
Usually, human pluripotent stem cells can be maintained and cultured in an undifferentiated state by co-culturing with feeder cells such as mouse embryonic fibroblast (MEF) or culturing in feeder cell-conditioned medium (CM). When human pluripotent stem cells are co-cultured with feeder cells or cultured in feeder cell-conditioned medium, unfortunately, there is the risk of cross-transfer of one or more pathogens such as virus from the xenogeneic feeders.
Recently, many studies have been made to develop a method for culturing human pluripotent stem cells without feeder cells and with defined factors only. To achieve this, it is important to develop methods of maintaining and proliferating stem cells in an undifferentiated state. The Xu group (Nat Biotechnol 18:399, 2000) established a feeder-free culture system using mouse embryonic fibroblast-derived CM and matrices such as laminin and matrigel. Further, Rosier (Bev Dyn 229:259, 2004) succeeded in maintaining human embryonic stem cells in a feeder-free condition for one year or longer by taking advantage of the CM established by the Xu group. However, such a CM suffers from a disadvantage in that a mouse-derived component is retained in the medium. In 2005, Xu et al. reported that undifferentiated human stem cells can be maintained in the absence of feeder cells by using bFGF in combination with other growth factors (STEM CELLS 23:315, 2005) or by adding an inhibitor of bFGF and BMP signaling pathway, noggin to the culture media without a conditioned medium (CM). It was also reported by Sato et al. that undifferentiated human embryonic stem cells could be maintained through the activation of the Wnt pathway using the GSK-3-specific inhibitor BIO in the absence of feeder cells (Nat. Med. 10:55, 2004).
Since most embryonic stem cells derived from the inner cell mass are either left in frozen storage or destroyed, there are no legal problems. However, due to the fact that extraction of stem cells from an embryo may be considered to be the destruction of human life, the debate over stem-cell research incorporates ethical and religious considerations. Moreover, since stem cells are derived from embryos in limited supply, immune incompatibility between individuals can cause immune rejection of transplanted cells in cell therapy. One of the alternatives to overcome these problems is that induced pluripotent stem cells (iPSC) having properties similar to embryonic stem cells are produced from somatic cells by the use of dedifferentiation factors (Cell 126, 663-676, 2006; Cell 131, 861-872, 2007; Nature 441, 1061-1067, 2006; Nature 451, 141-146, 2008). This success is expected to improve technology for practical use of pluripotent stem cells in stem cell therapy. Induced pluripotent stem cells are advantageous in that the generation of induced pluripotent stem cells does not require embryos, and the use of cells extracted from a patient does not cause immune rejection, and thus it becomes a very valuable tool for practical use. To advance the present technology to practical levels, it is important to develop follow-on technologies which improve the accompanying disadvantages, namely, low dedifferentiation efficiency and tumorigenic potential due to the use of integrating viruses for iPSC induction.
In this connection, some methods for improving dedifferentiation efficiency are reported to control extracellular conditions or use supplements, in particular, small molecule compounds. Further, it was also reported that dedifferentiation efficiency is effectively increased under hypoxic conditions similar to those of embryonic stem cells (Cell Stem Cell, 5: 237-241, 2009). Dr. Ding's group (Shi et al., Cell Stem Cell, 2008) reported that small molecule compounds such as BIX-01294 (G9a histone methyltransferase inhibitor), BayK8644 (L-type calcium channel agonist), and RG108 (DNA methyltransferase inhibitor) are effective for enhancing dedifferentiation efficiency and Dr. Melto's group (Huangfu et al., Nat Biotechnol, 2008) reported that small molecule compounds such as VPA (histone deacetylase inhibitor), TSA (histone deacetylase inhibitor), and SAHA (histone deacetylase inhibitor) are effective for enhancing dedifferentiation efficiency. There are suggested alternatives to the use of a virus: 1) transient expression of a single nonviral polycistronic vector (Gonzalez et al, PNAS USA, 2009; Chang at al, Stem cells, 2009), 2) application of an adenovirus (Stadtfeld at al, Science 2008), and 3) Cre/loxP recombinant expression control system (Soldner et al, Cell, 2009), iPSC establishment using a single nonviral polycistronic vector and removal of dedifferentiation cassette by Cre transfection (kaji at al, Nature, 2009), 4) piggyback (PB) transposon system (Woltjen et al, Nature, 2009; kaji et al, Nature, 2009), and 5) nonintegrating episomal vectors (Yu et al, Science, 2009). Nevertheless, there still remain the problems of genetic abnormalities and tumorigenic potential.
Neuropeptide Y (NPY) is a 36 amino-acid peptide which, together with pancreatic polypeptide (PP), belongs to a family of neuroendocrine peptides. The peptide is widely distributed throughout the central and peripheral nervous system of mammals, and in particular, is abundant in the hypothalamus and cerebral cortex. It is known that NPY exerts a remarkably wide variety of physiological effects of potential therapeutic importance, and induces vasoconstriction when administered alone, and can cause angina pectoris (Clarke, et al., Lancet 1(8541):1057 (1987)). In addition, NPY, which is a neurotransmitter distributed throughout the central and peripheral nervous system, stimulates appetite and decreases energy expenditure during starvation. For example, when injected into the brain, NPY increases appetite in various species, and chronically causes an increase in body weight and insulin resistance. In particular, NPY modulates leptin actions in the hypothalamus. Leptin- and leptin receptor-deficient rodents have increased hypothalamic NPY, whereas NPY deficiency makes leptin-deficient mice less obese. Even though NPY has such a variety of physiological actions, there have been no studies on the actions and use of NPY in human pluripotent stem cells.